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Large Scale Targeted Protein Quantification Using HR/AM Selected Ion Monitoring with MS/MS Confirmation on a Novel Hybrid, Q-OT-qIT Mass Spectrometer

Authors: Reiko Kiyonami1, Michael Senko1, Vlad Zabrouskov1, Jarrett Egertson2, Sonia Ting2, Michael MacCoss2, Andreas FR Hühmer1, José Felipe Lugão3

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1Thermo Fisher Scientific, San Jose, CA, USA; 2University of Washington, Seattle, WA, USA; 3Nova Analítica, São Paulo, SP, BR

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Proteomics studies are turning from qualitative to quantitative to understand the biological function and interactions of proteins in biological systems. The extreme complexity and dynamic range of proteins in typical samples challenges traditional data dependent workflows by requiring very fast MS/MS acquisition to reproducibly and deeply interrogate the sample. Recently, several data independent acquisition approaches1 have been explored to increase reproducibility and comprehensiveness for better quantification. These approaches use targeted extracted fragment ions from HR/AM MS/MS data collected with a wide isolation window such as 25 Da for quantification. The quantitative performance could be potentially compromised by interfering fragments from co-eluted background compounds2.
In this work a highly sensitive and selective data independent acquisition (DIA) workflow was developed for large-scale targeted protein quantification on a new mass spectrometer based on a mass resolving quadrupole, Orbitrap analyzer, collision cell, linear ion trap (Q-OT-qIT) architecture.
For data independent acquisition set-up, three high-resolution, accurate-mass (HR/AM) selected ion monitoring (SIM) scans (240,000 FWHM) with wide isolation windows (200 amu) were used to cover all precursor ions of 400 – 1000 m/z. In parallel with each SIM scan, 17 sequential ion trap MS/MS with 12 amu isolation windows were acquired to cover the associated 200 amu SIM mass range. Quantitative information for all precursor ions detected in three sequential SIM scans is recorded in a single run. Plus, all MS/MS fragment information over the mass range of 400 – 1000 m/z is recorded for sequence confirmation of any peptide of interest by querying specific fragment ions in the spectral library. The quantitative performances and throughput of this new approach were evaluated using various samples.
The data collected from SIM scans with high resolving power provided unambiguous detection of targeted peptide peaks even at low concentration levels by separating interferences from matrix from peptide associated signal. Ten (10) attomole limits of detection (LODs) were observed for the isotopically labeled standards spiked in 500 ng E. coli digest matrix. Four (4) orders of linear dynamic range were observed with good precision. Highly reproducible and complete quantitative results were achieved by applying a targeted data extraction strategy after the data independent acquisition.


  1. Gillet, L.C.; Navarro, P.; Tate, S.; Roest, H.; Selevsek, N.; Reiter, L.; Bonner, R.; Aebersold, R. Mol Cell Proteomics, 2012, as Manuscript O111.016717.
  2. Gallien. S.; Duriez. E.; Demeure. K.; Domon. B. Journal of Proteomics, 2013, 81, 148–158.